Abstract: In this ear of large-scale proteomic analysis it is apparent that proteins carry out cellular processes as members of dynamic multi-protein assemblies rather than simply working as isolated individuals. Technical advances in epitope tagging and mass spectrometry, as well as the use of genome-wide two-hybrid screens, have led to an explosion of data listing and mapping numerous protein-protein associations found with the cell. Although progress has been made cataloging the constituents of specific complexes, our understanding of how proteins assemble into higher order structures and how multi-protein complexes perform their cellular functions remains a significant challenge. Structural analysis of these proteins assemblies will be required to understand their organization and function. Single particle cryo-electron microscopy is a powerful structural technique that is uniquely suited for working with large dynamic complexes that are too difficult to crystallize. The spliceosome is a macromolecular machine that catalysis the excision of non-coding introns from a pre-messenger RNA (pre-mRNA). It is formed from five small nuclear ribonucleoprotein subunits (snRNPs) and numerous non-snRNP splicing factors. However, how the snRNPs are organized within a larger unit to execute the catalytic steps of pre-mRNA splicing is not known. Understanding how the spliceosome functions is important because many human diseases and some forms of cancer arise from pre-mRNA splicing errors. However, the large size and dynamic nature of spliceosomal complexes has made structural characterization extremely challenging. The goal of this proposal is to take an innovative multi-disciplinary approach to probe the structure and function of spliceosomal complexes. We will use cryo-EM in combination with yeast genetics and biochemistry to improve our understanding of how these very large, dynamic cellular machines are structurally organized and how this organization translates into function within the cell.